The invention relates to cell biology, genetics, recombinant DNA technology, fluorescence microscopy, and videography.
Messenger RNA localization is a well-documented phenomenon and provides a mechanism by which to generate cell assymetry (St. Johnston, Cell 81:161-170 (1995); Glotzer et al., Cell Dev. Biol. 7:357-365 (1996); Steward et al., in mRNA Metabolism and Posttranscriptional Gene Regulation, Wiley-Liss, New York, 127-146). Messenger RNA localization has been studied by fluorescence in situ hybridization (FISH) (Long et al., RNA 1:1071-1078 (1995). In situ hybridization, and other methods that require fixation of cells, offer good spatial resolution, but are severely limited in temporal resolution. Thus, while these techniques are well-suited for determining where RNA goes in living cells, they are unsuited for determining how quickly, or by what route, the RNA travels to its destination.
A further limitation of FISH methods, is that fixation kills cells. Therefore, those methods are incompatible with cell selection, where cells must be kept alive to initiate a new cell line.
We have developed a general method for visualizing the location and movement of a specific RNA of interest in a living cell, in real time. The method includes the following steps: (a) providing a DNA encoding the RNA, which RNA includes a protein-binding site; (b) providing a nucleic acid encoding a fusion protein that includes a fluorescent domain and an PNA-binding domain that binds to the protein-binding site in the RNA; (c) introducing the DNA encoding the RNA, and the nucleic acid encoding the fusion protein, into a eukaryotic cell so that the DNA encoding the RNA and the nucleic acid encoding the fusion protein are expressed in the cell; and (d) detecting fluorescence in the cell, the fluorescence being from the fusion protein bound to the RNA.
Preferably, the RNA includes a multiplicity of protein-binding sites located in the 3xe2x80x2 untranslated region (3xe2x80x2UTR) of the RNA. The RNA-binding domain can be derived from a bacteriophage MS2 protein, and the protein-binding site can be a bacteriophage MS2 binding site. In preferred embodiments of the invention, the fluorescent domain is derived from green fluorescent protein (GFP). In some embodiments, the fusion protein includes an intracellular localization domain, e.g., a nuclear localization signal (NLS) domain or a nuclear export signal (NES) domain. When the fusion protein contains an NLS domain, fluorescence from the fusion protein bound to the RNA is detected outside the nucleus.
The DNA encoding the RNA, and the nucleic acid encoding the fusion protein, can be provided on a single vector or on separate vectors. In some embodiments of the invention, the cell is a yeast cell. The cell can contain one or more RNA localization factors, e.g., she gene products in a yeast cell.
The invention also provides a method for screening a DNA library to detect a DNA encoding an RNA containing a protein-binding site. The method includes providing a eukaryotic test cell. The test cell expresses a fusion protein containing a fluorescent domain and an RNA-binding domain that binds to a protein-binding site. The method further includes transforming the test cell with a candidate DNA from the DNA library; and detecting the fusion protein bound to an RNA containing the protein-binding site, if present, by measuring fluorescence. In some embodiments, the fusion protein includes an intracellular localization domain, e.g., a nuclear localization signal (NLS) domain or a nuclear export signal (NES) domain. Preferably, the test cell does not express an endogenous protein that binds to the protein-binding site.
The invention also provides a nucleic acid encoding a fusion protein. The fusion protein encoded contains a fluorescent domain and an RNA-binding domain. The fluorescent domain can be derived from GFP or a GFP variant, e.g., blue fluorescent protein (BFP), yellow fluorescent protein (YFP), or cyan fluorescent protein (CFP). The binding domain can be derived from a bacteriophage MS2 binding protein. In some embodiments, the fusion protein includes an intracellular localization domain, e.g., a nuclear localization signal (NLS) domain or a nuclear export signal (NES) domain. The invention also includes a vector containing the nucleic acid encoding the fusion protein, and a cell transformed with the vector containing the nucleic acid encoding the fusion protein.
The invention also includes a screening method for identifying a compound that inhibits nuclear RNA export or import. The method includes providing a eukaryotic test cell that expresses a DNA encoding an RNA, which RNA includes a protein-binding site; and expresses a fusion protein. The fusion protein includes a fluorescent domain and an RNA-binding domain that binds to the protein-binding site in the RNA. The method further includes contacting the test cell with a candidate compound, and then detecting a candidate compound-related reduction of nuclear RNA export or import, if present. In some embodiments of the method, the RNA-binding domain of the fusion protein and the protein-binding site in the RNA are derived from viral sequences.
The invention also includes a method for detecting, in real-time, the transcription of a specific gene. The method includes providing a eukaryotic cell that contains: a DNA encoding an RNA that includes a protein-binding site, and a nucleic acid encoding a fusion protein. The fusion protein includes a fluorescent domain and an RNA-binding domain that binds to the protein-binding site. The method further includes detecting a focus of fluorescence, the focus being from a multiplicity nascent RNA molecules, each nascent RNA molecule being bound to one or more fusion protein molecules. In some embodiments, the fusion protein includes a nuclear export signal domain. The nucleic acid encoding the fusion protein can be transiently expressed from a vector introduced into the cell.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present application, including definitions will control. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference.
Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described below. The materials, methods, and examples are illustrative only and not intended to be limiting. Other features and advantages of the invention will be apparent from the detailed description, and from the claims.